浙江省某猪场暴发Ⅱ型猪链球菌感染及病原诊断和药敏试验

报告2003年秋季浙江省某猪场Ⅱ型猪链球菌感染猪群的微生物学检查和药物敏感试验结果,以及采取的控制疫情措施及其效果.从112头病、死猪中分离出71株猪链球菌疑似菌株,经革兰染色镜检、生化反应和特异性诊断血清的玻片凝集,均鉴定为Ⅱ型猪链球菌.上述Ⅱ型猪链球菌分离株对阿莫西林等5种抗生素敏感,但对庆大霉素等7种抗生素耐药.采取饲养环境和饲料消毒、发病猪隔离、阿莫西林和环丙沙星治疗、未发病猪疫苗接种等措施,有效地控制了疫情,未发生人的感染.     

广西、四川两地屠宰场猪链球菌致病特征分析

【目的】猪链球菌(Streptococcus suis)是猪的重要致病菌，同时也是一种人畜共患病原。屠宰场健康猪的猪链球菌感染率较高，血清型多而复杂，是人和猪的重要传染源。四川、广西两地都曾暴发猪链球菌疫情，大量生猪发病死亡的同时，也导致部分与病猪有密切接触的人群发病死亡。因此，对上述两地区屠宰场进行猪链球菌感染调查，明确其致病特征，具有重要公共卫生意义。【方法】本研究自2021-2022年采集广西、四川两地屠宰场健康猪扁桃体，分离鉴定猪链球菌并进行血清型分型，对分离株进行斑马鱼毒力、小鼠毒力试验，并对毒力株进行基因组测序，进行多序列位点分型(multilocus sequence typing, MLST)及毒力标志基因分析，探究其致病特征。【结果】广西57份健康猪扁桃体样品猪链球菌阳性率为84.21%(48/57)，分离鉴定出60株猪链球菌，其中分离率最高的是血清31型(16.67%, 10/60)，其次为9型(11.67%, 7/60)、4型(10.00%, 6/60)、12型(8.33%,5/60)等，2型菌株仅分离出1株，含2种及以上不同血清型猪链球菌的扁桃体占33.33%(...     

浙江某市屠宰场猪链球菌血清型、耐药及致病特征

【目的】猪链球菌(Streptococcus suis)是猪的重要病原菌,同时也是人畜共患病原。猪的扁桃体是猪链球菌主要定殖部位之一,是易感猪和人的重要传染源。因此,对屠宰场健康猪进行猪链球菌流行病学调查,具有重要的公共卫生学意义。【方法】本研究自2020年至2021年,从浙江某市屠宰场采集健康猪扁桃体样品,分离鉴定猪链球菌,采用血清型特异性PCR法分型,通过耐药基因检测、药敏试验、斑马鱼毒力实验分析其耐药及致病特征。【结果】131份健康猪扁桃体样品猪链球菌阳性率为62.59%(82/131),共分离猪链球菌68株,其中16型分离率最高,占比16.18%(11/68),其次为31型(11.76%,8/68)、9型(7.35%,5/68)、3型(7.35%,5/68)等。含2种及以上血清型的扁桃体样品占15.85%(13/82)。药敏试验表明,分离株主要对林可酰胺类(100%,68/68)、大环内酯类(98.53%,67/68)、四环素类(100%,68/68)抗生素耐药,所有菌株均属于多药耐药。值得关注的是,有18株菌对青霉素耐药、3株菌对头孢噻肟耐药、2株菌对利福平耐药、11株菌对利...     

四川绵阳地区肺炎链球菌流行病学特征

目的了解四川绵阳地区肺炎链球菌的耐药情况,为临床合理用药及感染控制提供依据。方法收集我院2015年1月至2017年12月临床分离的718株肺炎链球菌,并对其药物敏感性检测结果进行分析。结果肺炎链球菌对红霉素和四环素的耐药率最高,均在95%以上;对左氧氟沙星、莫西沙星、泰利霉素、氯霉素的敏感性较高,均在89%以上;对青霉素、美罗培南、阿莫西林和头孢类抗生素的耐药率均在30%以上;未检出万古霉素和利奈唑胺耐药株。肺炎链球菌主要耐药模式为头孢曲松+红霉素+四环素+复方新诺明+美罗培南+青霉素+阿莫西林+头孢噻肟,占19.44%。2017年肺炎链球菌青霉素敏感株和不敏感菌株对美罗培南、阿莫西林、厄他培南、头孢噻肟、头孢曲松、氧氟沙星、复方新诺明和氯霉素的不敏感率比较差异有统计学意义(P0.05)。结论本地区肺炎链球菌对红霉素、四环素耐药率较高,不适用于肺炎链球菌感染的治疗;对青霉素、美罗培南、阿莫西林和头孢类抗生素不敏感率较高,应慎重用于经验治疗;对万古霉素、利奈唑胺和喹诺酮类药物敏感性较高,临床可合理使用。     

猪链球菌2型毒力因子研究进展

猪链球菌病是由猪链球菌引起的一种重要的人善共患病.其中,猪链球菌2型不仅能够引起猪发病,使病猪主要表现为败血症、脑膜炎和关节炎;还可感染人,从而导致人脑膜炎和败血症,甚至死亡.该病严重威胁着人类的公共卫生安全,引起人们的高度重视.一般研究认为猪链球菌的致病性与其毒力因子有密切关系,本文着重对猪链球菌2型毒力因子进行阐述.     

安徽PRSS发病猪副猪嗜血杆菌的分离鉴定及其耐药性分析

目的了解猪繁殖与呼吸综合征（PRRS）发病猪继发感染副猪嗜血杆菌（HPS）的情况及分离菌株的药物感受性。方法应用细菌分离培养、形态学检查、生化试验和PCR技术,对2007年1月至2008年12月从安徽不同地区采集的146头份PRRS发病猪的病料进行HPS检测,并采用标准K-B纸片法对分离菌株进行14种抗菌药物敏感试验。结果分离鉴定出12株HPS,检出率为8.22%（12/146）;12株HPS对氯霉素100%敏感,环丙沙星为91.7%,阿莫西林和新霉素为83.3%,对罗红霉素100%耐药,阿米卡星为83.3%。结论安徽省不同地区PRRS感染猪群中均存在程度不同的HPS感染,各地区HPS分离株表现出形态上的变化特征和一致的生化特性,且有对临床常用抗菌药物耐药性增强的趋势。     

致病性大肠杆菌和蜡样芽胞杆菌的分离鉴定

【背景】猪只消化道疾病是养猪业上一大重要疾病,给养猪业带来一定的经济损失。大肠杆菌是引起猪腹泻的一种常见病原菌,可以引起不同日龄的猪腹泻,但主要以幼龄猪为主。【目的】旨在分离鉴定引起四川省眉山市一规模化养猪场病猪大规模腹泻的病原菌。【方法】采用常规细菌分离方法结合16S rRNA基因序列的分析方法从发病猪肝脏、胃以及污染的饲料分离鉴定细菌,并对分离株进行小鼠致病性试验、16S rRNA基因遗传进化树分析、毒力基因的检测、药物敏感试验。【结果】从腹泻猪肝脏中分离到一株致病性大肠杆菌,胃中分离到一株蜡样芽胞杆菌,并且追溯到传染源是该猪场饲料。通过检测这两株菌相应的毒力基因发现大肠杆菌不属于肠外致病性型,蜡样芽胞杆菌检测到了nheA、nheB、nheC、bceT、entFM 5种毒力基因;药敏试验表明常规的氨基糖苷类和头孢类抗生素对大肠杆菌抑菌效果较好,红霉素、氟苯尼考、头孢氨苄、头孢哌酮对蜡样芽胞杆菌抑菌效果较好,而蜡样芽胞杆菌对青霉素、阿莫西林等常规药物不敏感。【结论】饲料存在大肠杆菌和蜡样芽胞杆菌的混合污染。     

218例表皮葡萄球菌的耐药性分析

目的了解3年内218例表皮葡萄球菌的耐药性,指导临床合理使用抗生素,方便临床采取更有效的治疗措施。方法对2007年1月至2009年12月间盛泽医院218例感染患者的各个感染部位进行表皮葡萄球菌的分离和耐药性检测,根据药物敏感试验结果来分析其耐药性的变化。结果 218株分离的表皮葡萄球菌中,耐甲氧西林菌株(MRSE)160株,占总数的73.4%,甲氧西林敏感菌株(MSSE)58株,占26.6%,未发现对万古霉素耐药的菌株,MRSE对大部分抗生素有较高的耐药率,MSSE则对大多数抗生素保持较低的耐药率。结论表皮葡萄球菌的分布广泛,耐药情况日趋严重,合理使用抗菌药物以延缓细菌耐药性的产生非常重要。     

一株分离自流浪猫的猪链球菌9型的分子生物学鉴定北大核心CSCD

【目的】猪链球菌是一种能感染人和猪的人畜共患病病原,并且还可零星感染多种哺乳动物。本试验旨在调查流浪猫携带猪链球菌的情况。【方法】在流浪猫身上分离猪链球菌,经血清凝集实验和PCR检测,鉴定其血清型;经多序列位点分型分析,鉴定其ST型;将所分离的细菌与Gen Bank上已公布的猪链球菌构建16S rRNA的系统发育树,分析该菌株与其他猪链球菌的亲缘关系;药敏纸片法分析其耐药性;小鼠攻毒试验分析其毒力。【结果】本试验在流浪猫身上分离到一株猪链球菌,命名为m70,其血清型为9型。多序列位点分型显示,m70株属于一个新的ST型。与Gen Bank上已公布的猪链球菌16S rRNA进行系统发育树分析,结果显示m70属于一个单独的分支。m70与临床菌株的耐药情况相似,对四环素耐药,对红霉素中介耐药,对氨苄西林敏感。小鼠攻毒试验显示,感染10~8 CFU剂量m70的小鼠,死亡率达到60%–80%（3/5–4/5）,3次攻毒试验的平均LD（50）为5.1×107 CFU;而本实验室保存的猪链球菌强毒株HA9801感染小鼠的平均LD（50）为3.9×107 CFU,两者之间没有显著差异（P〈0.05）。【结论】从流浪猫身上分离得到的猪链球菌m70属于优势血清型,且毒力较强,提示一些流行血清型的猪链球菌强毒株具有从流浪猫传染人的潜在风险。     

猪链球菌2型05Z33强毒株转录调控因子Rgg基因敲除突变体的构建及毒力分析

目的:构建猪链球菌2型强毒株05ZYH33转录调控因子Rgg的基因敲除突变体,观察其生物学性状,并在动物感染实验中比较敲除株与野生株的毒力差异,为进一步研究猪链球菌转录调控因子在致病中的作用提供实验基础。方法:分别以猪链球菌2型05ZYH33基因组和pSET1质粒为模板,扩增基因SSU05_1997两侧各约500 bp的片段为上下游同源臂,氯霉素(Cm)抗性基因为中间片段,采用重叠PCR方法连接3个片段;连接产物先克隆到T载体上,再经过酶切克隆到温度敏感自杀载体pSET4S上;将构建的基因敲除载体pSET4S-1997电转化入05ZYH33感受态细胞,通过改变培养温度筛选出基因敲除突变体05Z33△rgg;对敲除株和野生株的生物学性状及小鼠和猪的致病性进行了初步比较。结果:PCR分析和测序结果均显示基因SSU05_1997完全被Cm抗性基因所替代,基因敲除突变体构建成功;05ZYH33△rgg对小鼠和猪的致病性与野生株相比无明显差异。结论:转录调控因子Rgg可能和猪链球菌2型的毒力无关。     

Application of a bacteriophage lysin to disrupt biofilms formed by the animal pathogen Streptococcus suis

Bacterial biofilms are crucial to the pathogenesis of many important infections and are difficult to eradicate. Streptococcus suis is an important pathogen of pigs, and here the biofilm-forming ability of 32 strains of this species was determined. Significant biofilms were completely formed by 10 of the strains after 60 h of incubation, with exopolysaccharide production in the biofilm significantly higher than that in the corresponding planktonic cultures. S. suis strain SS2-4 formed a dense biofilm, as revealed by scanning electron microscopy, and in this state exhibited increased resistance to a number of antibiotics (ampicillin, amoxicillin, ciprofloxacin, kanamycin, and rifampin) compared to that of planktonic cultures. A bacteriophage lysin, designated LySMP, was used to attack biofilms alone and in combination with antibiotics and bacteriophage. The results demonstrated that the biofilms formed by S. suis, especially strains SS2-4 and SS2-H, could be dispersed by LySMP and with >80% removal compared to a biofilm reduction by treatment with either antibiotics or bacteriophage alone of less than 20%; in addition to disruption of the biofilm structure, the S. suis cells themselves were inactivated by LySMP. The efficacy of LySMP was not dose dependent, and in combination with antibiotics, it acted synergistically to maximize dispersal of the S. suis biofilm and inactivate the released cells. These data suggest that bacteriophage lysin could form part of an effective strategy to treat S. suis infections and represents a new class of antibiofilm agents.     

Streptococcus Suis Isolated from Pigs in Finland

A total of 58 Streptococcus suis strains were isolated from deceased pigs submitted to the National Veterinary Institute, Regional Laboratory in Kuopio, Finland, over a 3 1/2 year period, most frequently from cases of pneumonia. The bacteria were isolated from cases of meningitis, sepsis, rhinitis, endocarditis and abortion. S. suis was also isolated from nasal cavity, lung and brain of some sick piglets without signs of inflammation. Further S. suis was detected in 12 out of 107 tonsils of healthy fatteners tested. S. suis strains were identified by biochemical methods followed by typing. The most common capsular types were 7, 3 and 2, respectively. Only one type 1 strain and no types 6 and 9 strains were found. All S. suis strains tested were sensitive to penicillin and ampicillin. S. suis is not uncommon in Finnish pig herds. S. suis may be regarded as a potentially pathogenic organism which under certain predisposing conditions may cause serious disease.     

猪链球菌2型荚膜唾液酸对细菌毒力和宿主炎症反应的影响

【目的】阐明猪链球菌2型荚膜唾液酸是否影响细菌毒力以及宿主对其炎症反应应答,为研究猪链球菌2型的致病机制奠定基础。【方法】比较实验菌株对BLAB/c小鼠模型的致病性;通过涂板计数的方法检测实验菌株在小鼠体内的分布;观察小鼠脑组织病理改变,分析实验菌株感染小鼠后中枢神经系统的病变差异;从小鼠体外全血细胞水平,运用ELISA法检测实验菌株感染后细胞炎性因子的分泌水平。【结果】荚膜唾液酸合成基因neuB缺失突变株ΔneuB相比野生株05ZYH33株,对小鼠毒力显著降低,回复突变株cΔneuB毒力回复至野生株水平;野生株和突变株在血液及脑组织中分布具有显著差异,均可致BLAB/c小鼠脑组织不同程度的损伤;与野生株组相比较,细菌/细胞相互作用不同时间点后,突变株组体外刺激小鼠全血细胞分泌MCP-1、IL-6的水平显著提高;【结论】荚膜唾液酸影响细菌的毒力及宿主细胞对其的炎症反应应答,它是猪链球菌2型穿透血脑屏障导致脑膜炎的重要毒力因子。     

四川省资阳市爆发的猪链球菌感染疫情中病原菌的分离和鉴定

对四川资阳地区病猪标本、尸检肝标本和血清标本进行病原菌分离,得到10株分离菌。经菌落形态和菌体形态观察、生化鉴定,证明其中3株为猪链球菌2型(SS2)。通过对3株SS2胞外因子基因、溶菌酶释放蛋白基因、荚膜多糖基因和16S rRNA基因进行PCR扩增,结果分别在626bp8、85bp4、87bp和297bp处出现目的条带。为进一步了解分离的SS2菌株的特性,使用VITEK GPS-107药敏卡进行药敏试验,结果表明分离菌对青霉素-G、红霉素、万古霉素等多种抗生素敏感。分离菌感染Balb/c小鼠可引起动物死亡,并出现胃肠肿胀、嘴部青紫以及皮下紫斑等症状,与患者症状相似,小鼠脏器压片经革兰氏染色镜下观察可见阳性球菌。     

A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase

Polymerase chain reaction (PCR) primers that flank a 688-bp segment within the glutamate dehydrogenase gene (gdh) of Streptococcus suis type 2 could amplify efficiently the DNA of all 306 (100%) clinical S. suis isolates tested (pigs, n=305; human, n=1) encompassing all serotypes obtained from diverse organs, and geographic origins. When DNA from other bacteria were used as templates for amplification, no product was detected indicating specificity of the primers. Multiplex PCR was developed using the gdh gene primer pair and primers that targeted the gene encoding S. suis capsular biosynthesis (cps). This strategy enabled the detection of strains belonging to serotypes 1/2, 1, 2, 7, and 9, respectively. Using the multiplex-PCR technique, 12 out of 14 (86%) isolates that were previously identified as non-typable S. suis (based on biochemical reactions and serology) gave positive PCR results of which four were positive for serotype 7, three for serotype 2, and five for S. suis strains that belong to other serotypes. Retest results of all 14 isolates by several veterinary laboratories were identical with PCR and confirmed that the two non-PCR reactive isolates belonged to strains of other streptococcal species. These results indicated that PCR improved species determination and can thus be used as a reliable species-specific molecular diagnostic reagent for the accurate identification of S. suis isolates and a serotype-specific method for the detection of strains of serotypes 1/2, 1, 2, 7, and 9, respectively. The PCR method therefore has potential clinical and epidemiological applications.     

Differences in the population structure of invasive Streptococcus suis strains isolated from pigs and from humans in The Netherlands

Streptococcus suis serotype 2 is the main cause of zoonotic S. suis infection despite the fact that other serotypes are frequently isolated from diseased pigs. Studies comparing concurrent invasive human and pig isolates from a single geographical location are lacking. We compared the population structures of invasive S. suis strains isolated between 1986 and 2008 from human patients (N?=?24) and from pigs with invasive disease (N?=?124) in The Netherlands by serotyping and multi locus sequence typing (MLST). Fifty-six percent of pig isolates were of serotype 9 belonging to 15 clonal complexes (CCs) or singleton sequence types (ST). In contrast, all human isolates were of serotype 2 and belonged to two non-overlapping clonal complexes CC1 (58%) and CC20 (42%). The proportion of serotype 2 isolates among S. suis strains isolated from humans was significantly higher than among strains isolated from pigs (24/24 vs. 29/124; P<0.0001). This difference remained significant when only strains within CC1 and CC20 were considered (24/24 vs. 27/37,P?=?0.004). The Simpson diversity index of the S. suis population isolated from humans (0.598) was smaller than of the population isolated from pigs (0.765, P?=?0.05) indicating that the S. suis population isolated from infected pigs was more diverse than the S. suis population isolated from human patients. S. suis serotype 2 strains of CC20 were all negative in a PCR for detection of genes encoding extracellular protein factor (EF) variants. These data indicate that the polysaccharide capsule is an important correlate of human S. suis infection, irrespective of the ST and EF encoding gene type of S. suis strains.     

Prevalence of Streptococcus suis genotypes in wild boars of Northwestern Germany

Invasive serotype 2 (cps2+) strains of Streptococcus suis cause meningitis in pigs and humans. Four case reports of S. suis meningitis in hunters suggest transmission of S. suis through the butchering of wild boars. Therefore, the objective of this study was to investigate the prevalence of potentially human-pathogenic S. suis strains in wild boars. S. suis was isolated from 92% of all tested tonsils (n=200) from wild boars. A total of 244 S. suis isolates were genotyped using PCR assays for the detection of serotype-specific genes, the hemolysin gene sly, and the virulence-associated genes mrp and epf. The prevalence of the cps2+ genotype among strains from wild boars was comparable to that of control strains from domestic pig carriers. Ninety-five percent of the cps2+ wild boar strains were positive for mrp, sly, and epf*, the large variant of epf. Interestingly, epf* was significantly more frequently detected in cps2+ strains from wild boars than in those from domestic pigs; epf* is also typically found in European S. suis isolates from humans, including a meningitis isolate from a German hunter. These results suggest that at least 10% of wild boars in Northwestern Germany carry S. suis strains that are potentially virulent in humans. Additional amplified fragment length polymorphism analysis supported this hypothesis, since homogeneous clustering of the epf* mrp+ sly+ cps2+ strains from wild boars with invasive human and porcine strains was observed.     

Pro-inflammatory cytokine and chemokine release by human brain microvascular endothelial cells stimulated by Streptococcus suis serotype 2

Streptococcus suis serotype 2 is a world-wide agent of diseases among pigs including meningitis, septicemia and arthritis. This microorganism is also recognized as an important zoonotic agent. The pathogenesis of the meningitis caused by S. suis is poorly understood. We have previously shown that S. suis is able to adhere to human brain microvascular endothelial cells (BMEC), but not to human umbilical vein endothelial cells (HUVEC). The objective of this work was to study the ability of S. suis serotype 2 to induce the release of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1); IL-6 and the chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1) by human BMEC and HUVEC, using a sandwich enzyme-linked immunosorbent assay. S. suis was able to stimulate the production of IL-6, IL-8 and MCP-1 by BMEC but not HUVEC, in a time- and concentration-dependent manner. Bacterial cell wall components were largely responsible for such stimulation. The human and pig origin of strains does not seem to affect the intensity of the response; indeed, a very heterogeneous pattern of cytokine and chemokine production was observed for the different strains tested in this study. In situ production of cytokines and chemokines by BMEC may be the result of specific adhesion of S. suis to this cell type, with several consequences such as increased recruitment of leukocytes and an increase in the blood-brain barrier permeability.     

Studies on a murine model for evaluation of virulence of Streptococcus suis capsular type 2 isolates

Five different parameters, time of incubation of the culture, type of culture medium, inoculum, strain of inbred mice, and age of mice, were tested using the LD50 technique to standardize a murine model for the evaluation of the virulence of Streptococcus suis capsular type 2 isolates. A model using 28 day-old mice belonging to CF1 strain appeared to give the best results. The inoculum size was the parameter most influencing the 50% lethal dose obtained with mice. Inoculation with 1-ml volume of a bacterial suspension instead of 0.1 or 0.5 ml decreased the LD50. The standardized model was used to evaluate the virulence of some isolates of known pathogenicity for pigs. The minimum lethal dose was used in the model and it appeared that the virulence of Streptococcus suis capsular type 2 isolates can be measured from highly virulent to totally avirulent.     

Production and properties of bacteriocin-like inhibitory substances from the swine pathogen Streptococcus suis serotype 2

Streptococcus suis serotype 2 is a major pathogen found in the upper respiratory tract of swine. In this study, isolates of this bacterial species were tested for the production of bacteriocin-like inhibitory substances (BLIS). Of the 38 strains tested, four inhibited the growth of other S. suis isolates according to a deferred-antagonism plate assay. Interestingly, three of the strains were originally isolated from healthy carrier pigs and were considered nonvirulent. Three isolates (94-623, 90-1330, and AAH4) that produced BLIS in liquid broth were selected for further characterization. None of the inhibitory activities was related to the production of either organic acids or hydrogen peroxide. The BLIS produced by these strains were heat stable and proteinase K, pronase, and elastase sensitive but were trypsin and chymotrypsin resistant. They were stable at pH 2 and 12 and had molecular masses in the range of 14 to 30 kDa. Maximum production was observed during the mid-log phase. Following a curing procedure with novobiocin, only 90-1330 lost the ability to produce BLIS, suggesting that the BLIS might be plasmid encoded. Analysis of the inhibitory spectra revealed that the BLIS-producing strains also inhibited the growth of Actinobacillus minor, Actinobacillus porcinus, Enterococcus durans, Micrococcus luteus, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. dysgalactiae, Streptococcus equi subsp. zooepidemicus, and S. dysgalactiae subsp. equisimilis. This study reports for the first time the ability of the swine pathogen S. suis serotype 2 to produce BLIS with the characteristics of classic bacteriocins. Further studies are required to investigate the possibility of using bacteriocin-producing strains to prevent swine infections caused by virulent strains of S. suis serotype 2.